Of the five Plasmodium species known to infect humans, P. falciparum and P. vivax cause the most malaria cases worldwide, and thus pose the greatest public health challenge. Conducting comprehensive field studies, we discovered that both of these human pathogens have their origins in African apes (1-7). We found that chimpanzees and gorillas harbor at least six Plasmodium species that are closely related to P. falciparum (comprising the Laverania subgenus) as well as parasites that are nearly identical to human P. vivax (1-7). Phylogenetic analysis of ape-derived sequences showed that P. falciparum evolved following a single host switch of a gorilla parasite (1, 6), while P. vivax emerged from within a Plasmodium species that infects both chimpanzees and gorillas (3). Although the origins of the human pathogens are now well established, nothing is known about the evolutionary and mechanistic processes that led to their emergence; yet, such information is critical to understand how ape parasites crossed the species barrier and whether such events are likely to occur again. In this resubmission application, we propose to address these questions by characterizing the ape precursors of the human parasites at the whole genome level and by determining the species and host preferences of the Anopheles vectors that transmit these ape parasites. Our hypothesis is that comparative and population genomic studies of ape Plasmodium parasites, coupled with analyses of their transmitting mosquito vectors, will yield new insight into the biology of P. falciparum and P. vivax and reveal the processes that allowed ape parasites to colonize humans. One major obstacle has been that ape Plasmodium samples are virtually impossible to obtain due to the endangered species status of their hosts. In the past grant period, we have solved this problem by developing a selective whole genome amplification (SWGA) technique that generates -- from unprocessed ape blood and fecal samples as well as infected mosquitoes -- sufficient numbers of parasite genomes for nextgen sequencing. Using this novel technology, we have already sequenced several near full-length ape Plasmodium genomes, which revealed a horizontal gene transfer in the precursor of P. falciparum (RH5 locus) and a new reticulocyte binding protein gene (RBP-3) in ape P. vivax. We will use fecal and blood samples and infected mosquitoes to sequence the genomes of additional members of each of the six ape Laverania species (Aim #1) as well as chimpanzee and gorilla P. vivax parasites (Aim #2). Comparative genomic analyses will identify loci that are unique to ape versus human Plasmodium species as well as genes that have been subject to positive selection. These genetic studies will be complemented by field and laboratory studies of the Anopheles vectors that transmit ape Plasmodium parasites (Aim #3), as well as hypothesis driven mechanistic studies of host-parasite interactions (Aim #4). Execution of these aims will substantially advance our understanding of the pathways that led to the emergence of P. falciparum and P. vivax, and inform eradication efforts of future zoonotic risk.